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Full text of "Methods of testing and analyzing excito-repellency responses of malaria vectors to insecticides."

Journal of the American Mosquito Control Association, 13(1): 13-17, 1997 

METHODS OF TESTING AND ANALYZING EXCITO-REPELLENCY 
RESPONSES OF MALARIA VECTORS TO INSECTICIDES 1 

DONALD R. ROBERTS, 2 THEERAPHAP CHAREONVIRIYAPHAR 3 
HAROLD H. HARLAN 4 and PAUL HSHIEH 2 

ABSTRACT. A new test system that includes an excito-repellency test box, test procedures, and statistical 
treatment of data is described. The method consists of enclosing 25 mosquitoes in an exposure chamber lined 
with insecticide-treated or untreated (control) test papers. Each chamber has a single portal for mosquitoes to 
escape to a receiving cage, and numbers escaping are manually recorded at 1-min intervals. The exposure 
chamber accommodates a screened, 2nd chamber that, when placed in the exposure chamber, prevents the 
mosquitoes from making physical contact with test papers. A full assay utilized one exposure chamber that 
permits physical contact with insecticide-treated papers, one chamber that permits physical contact with control 
papers, one chamber that prevents physical contact with insecticide-treated papers, and a 4th chamber that 
prevents contact with control papers. After insecticide exposure, test populations are held for observations on 
24-h mortalities. A survival analysis approach is described for estimating mosquito escape rates and for com- 
paring differences in mosquito escape rates, with or without physical contact with insecticide, among populations, 
insecticides, and doses of insecticide. 



INTRODUCTION 

Assays for evaluating behavioral responses of 
malaria vectors to insecticide residues have been 
reviewed by Muirhead-Thomson (1960), Coluzzi 
(1963), Busvine (1964), and Elliott (1972). The test 
of greatest value for studies of insecticide avoid- 
ance was described by Coluzzi (1963) as a box with 
slits for escaping. Such a box was described by 
Rachou et al. (1973) and is referred to as the excito- 
repellency test box. Similar excito-repellency test 
boxes are described by Rachou et al. (1973), Charl- 
wood and Paraluppi (1978), Roberts et al. (1984), 
Rozendaal et al. (1989), and Evans (1993). In ex- 
cito-repellency tests, mosquitoes are released inside 
a box lined with sprayed paper. Outlets in the form 
of out-projecting baffles permit the mosquitoes to 
escape into 2 separate cages. The baffles prevent 
the mosquitoes from reentering the box and the 
numbers escaping are counted by time postrelease. 
The difficulties of working with test boxes were 
described by Roberts et al. (1984). Major problems 
relate to the difficulties in introducing specimens 
into the boxes, removing live specimens at the end 
of test periods, and providing a standardized insec- 
ticide dose. The lack of an appropriate method of 
data analysis has been another shortcoming of the 
test method. Earlier methods did not test for behav- 



1 This research was supported by grant R087EK from 
the Uniformed Services University of the Health Sciences, 
Bethesda, MD. The views of the authors do not purport 
to reflect the positions of the U.S. Department of Defense 
or the Uniformed Services University of the Health Sci- 
ences. 

2 Department of Preventive Medicine and Biometrics, 
Uniformed Services University of the Health Sciences, 
4301 Jones Bridge Road, Bethesda, MD 20814-4799. 

3 Kampheangsaen Campus, Kasetsart University, Fac- 
ulty of Liberal Arts & Sciences, Nakhon Prathom 73140, 
Thailand. 

4 Maple Hill Lane, Crownsville, MD 21032-1062. 



ioral responses without physical contact with insec- 
ticide-treated papers. 

Described herein are improved boxes for testing 
behavioral responses of adult Anopheles mosqui- 
toes with or without physical contact with insecti- 
cide residues. Survival analysis methods are de- 
scribed for the statistical treatment of test data. 



MATERIALS AND METHODS 

The test method consists of enclosing 25 mos- 
quitoes in an exposure chamber lined with insecti- 
cide-treated or untreated (control) papers. Each ex- 
posure chamber has a single portal for mosquitoes 
to escape to a receiving cage. The exposure cham- 
ber accommodates a screened, 2nd chamber (inner 
chamber) that, when placed in the first chamber, 
prevents the mosquitoes from making physical con- 
tact with test papers. Under test conditions, mos- 
quitoes are enclosed within the exposure chamber 
and the only source of light comes from the exit 
portal. A full assay consists of 4 exposure chambers 
of 2 treatment chambers and 2 control chambers, 
as shown in Table 1 . Treatment chambers are lined 
with test papers impregnated with insecticide and 
an oil-based carrier. Control chambers are lined 
with papers impregnated with carrier alone. One 
treatment chamber permits tarsal contact with in- 
secticide. The second treatment chamber includes 
the inner chamber, so mosquitoes cannot make tar- 
sal contact with insecticide. For brevity, tests with 
or without the inner chambers, for either treatment 
or control papers, are referred to as contact trials 
(no inner chamber) or noncontact trials (with an 
inner chamber). 

Components of the excito-repellency chambers 
are illustrated and numbered in Fig. 1. Except for 
a inner panel (No. 1), the exposure chamber is con- 
structed of metal and can be chemically cleaned. 
The exposure chamber (No. 4) is constructed of 
stainless steel and each chamber is 34 X 32 X 32 



13 



14 



Journal of the American Mosquito Control Association 



Vol. 13, No. 1 



Table 1 . Test conditions for evaluating behavioral 
responses of malaria vectors to insecticide residues. 



Papers lining the 
exposure chamber 


With or without 

contact 
with test papers 

With Without 


With insecticide 

(treatment chambers) 
Without insecticide 

(control chambers) 


X 

X 


X 
X 



cm. The front panel is 32 X 32 cm and is equipped 
with an escape portal (No. 6). The escape portal is 
an outward projecting funnel (exit funnel), 14.75 
cm at its base. The top and bottom of the exit fun- 
nel are 14 cm long and converge, leaving a 1.50- 
cm-wide opening (a horizontal slit) through which 
the mosquitoes can escape from the exposure 
chamber. The back of the exposure chamber is a 
hinged metal door (No. 5) that closes tightly. The 
exposure chamber is also equipped with an inner 
removable rear panel (No. 1). This panel fits inside 
the back of the exposure chamber, abuts 4 small 
flanges inside the chamber, and serves to imprison 



Rear View 



1: Transparent 
plexiglass panel, 
31 X 30.5 cm. 




2: Dental dam, sealed 

port (15.5 cm in dia.) for putting 

specimens inside the chamber. 

3: Screened inner chamber, 
28.5 X 28.5 X 29 cm. 



Escaping 
mosquitoes 
6 



4: Stainless steel exposure 

(outer) chamber, 34 X 32 X 32 cm. 



Front View 



5: Hinged, stainless steel rear door. 



6: Stainless steel escape louvre, 
slit was 1 .5 cm wide. 



Fig. 1. An excito-repellency test box for the study of behavioral responses of mosquitoes to insecticides. 



March 1997 



Excito-Repellency Testing 



15 



the test population inside the exposure chamber. 
Plexiglas® is used for the inner rear panel so mos- 
quitoes can be observed inside the chamber. The 
Plexiglas panel is equipped with a large round hole 
(15.5 cm in diameter) that is sealed with a split 
piece of dental dam (No. 2). This sealed opening is 
used for placing mosquitoes inside and for remov- 
ing mosquitoes from the chamber. The 2 rear panels 
fulfill several requirements. First, the test popula- 
tion must be in darkness so imprisoned specimens 
can orient on light filtering through the escape fun- 
nel; thus the chamber needs to be solid and non- 
transparent. This requirement is fulfilled by closing 
the rear metal door at the start of each test. Second, 
the investigator needs to see inside the chamber to 
check for dead versus live specimens, both pre- and 
posttesting, and to remove live specimens at the 
end of the test. The ability to see inside the chamber 
is fulfilled by using transparent Plexiglas for the 
inner rear panel. Third, a self-sealing portal is need- 
ed for placing a test population inside the chamber 
and for removing specimens from the chamber at 
the end of each test. This requirement is fulfilled 
by using a split dental dam seal on the 15.5-cm- 
diameter opening in the Plexiglas rear panel. 

The frame of the inner chamber (No. 3) is con- 
structed of 0.62 X 0.62-cm aluminum beams. The 
structure of each chamber is 28.5 X 28.5 X 29 cm 
and the inner surface of each is covered with metal 
screening. A fine mesh metal screen, 52 cells per 
inch, covers the top, bottom, and 2 side walls of 
the inner chamber. The inner chamber is open end- 
ed, with 0.62-cm rubber gaskets on the front and 
back beams. When placed in the outer chamber, the 
front gasket seals small gaps between the front 
stainless steel panel and the inner chamber. Like- 
wise, the rear gasket seals gaps between the Plexi- 
glas panel and the inner chamber. The inner screen 
surface is no closer than 0.62 cm from the surface 
of test papers, and it prevents mosquitoes from 
making tarsal contact with the surface of test pa- 
pers. 

The receiving cage is a one-gal (1.6-liter) ice 
cream carton with a screened top. The cage fits over 
the outward projecting exit funnel. A section of or- 
thopedic stocking is attached to an opening in the 
side of the carton. The funnel of the exposure 
chamber is inserted through the stocking and 
through the opening of the receiving cage. 

Test papers lining the exposure chambers are first 
clipped, with metal paper clips, to large sheets of 
clean white typing paper. The large papers are taped 
together in a ribbon effect. Then, with test papers 
attached, the ribbon of paper is placed against the 
sides, top and bottom of the exposure chamber. Pa- 
pers are secured to the walls by pairing a small 
magnet on outside of the wall with a paper clip on 
the inside wall, the paper is secured by attraction 
between the magnets and paper clips. Test papers 
are not positioned on the front or back of the ex- 
posure chamber. 



A full test requires 4 groups of 25 mosquitoes 
(test population) each. With 2 investigators, test 
populations can be introduced into each of the 4 
exposure chambers in approximately 1 min. Before 
mosquitoes are introduced into the exposure cham- 
bers, exit funnels are sealed with Styrofoam® in- 
serts. A 3 -min rest period has been used to permit 
mosquitoes to adjust to test chamber conditions in 
other test procedures (Busvine 1964); therefore, a 
3-min interval is used in the present procedure. Af- 
ter 3 min, the Styrofoam insert is removed from 
each of the escape funnels to initiate the observa- 
tion period. Numbers escaping from exposure 
chambers to receiving cages are recorded manually 
at 1-min intervals; after 5 min of observation, re- 
ceiving cages are replaced with clean cages. The 
exchange of receiving cages facilitates the accurate 
counting of numbers escaping for each time inter- 
val. 

A survival analysis approach is used to estimate 
the rates of mosquitoes escaping from chambers. In 
the excito-repellency test, there are only 2 possible 
outcomes for a specimen: it will either escape or 
not escape from the exposure chamber. Binary test 
data are optimized for survival analysis techniques 
by only working with counts of specimens that do 
not escape. However, an estimate of escape rate or 
probability of escape is obtained by subtracting 
from one the estimated rate or estimated probability 
of remaining in the exposure chamber. These sta- 
tistically defined estimates for 1-min observation 
periods can be used to compare differences in mos- 
quito escape rates among populations, insecticides, 
and concentrations (doses) of insecticides, in either 
contact or noncontact trials. The analytical results 
can be presented either as proportions escaping or 
proportions remaining in exposure chambers. 

In this analysis, mosquitoes that escape are treat- 
ed as "deaths" and those remaining in the exposure 
chamber from one minute to the next as "surviv- 
als." Specimens in the exposure chamber at the end 
of the test are treated as "censored." In survival 
analysis terminology, the survival time of a speci- 
men is thought to be censored when the end point 
of interest (in our case, escape from the exposure 
chamber) has not been observed for that specimen 
(Lee 1992, Collett 1994). Time (min) for 50% and 
90% of the test population to escape is estimated 
with the life table method, and these estimates are 
used as "escape time" summary statistics (ET^ and 
ET„). 

The log-rank method is used to compare patterns 
of escape behavior (analogous to survival curves). 
This test is designed to detect differences between 
survival curves that result when the death (or es- 
cape) rate in one group is consistently higher than 
the corresponding rate in a 2nd group and the ratio 
of these 2 rates is consistent over time (in survival 
analysis, this is also called the proportional hazard 
rate). With excito-repellency data, the basic idea 
underlying the log-rank test involves examining es- 



16 



Journal of the American Mosquito Control Association 



Vol. 13, No. 1 



cape observations by 1-min intervals. To test the 
null hypothesis, we calculate the observed escape 
and expected escape in each 1-min interval. The 
data are analyzed by use of tabular data presenting 
columns for time, number observed to escape, 
number expected to escape, and difference between 
observed and expected. We then combine the tab- 
ular data for each test to give an overall measure 
of the deviation of the observed escape values from 
their expected values by each 1-min test interval. 
The log-rank method was proposed by Mantel and 
Haenzel (1959); it is also called the Mantel-Cox 
and Peto-Haenzel methods (Mantel and Haenzel 
1959). 

The log-rank test has a chi-square distribution 
with k degrees of freedom, where k is the number 
of groups- 1. A statistical software package, STA- 
TA® 5 , can be used for this analysis to test for dif- 
ferences among or between populations, dose lev- 
els, and insecticides. 

RESULTS AND DISCUSSION 

The World Health Organization's (WHO) rec- 
ommended tests of malaria vectors for behavioral 
responses to insecticides do not discriminate be- 
tween contact versus noncontact stimulation (WHO 
1975). The tests are based on the concept that ma- 
laria vectors respond to insecticides only after 
physical contact with the chemical and this concept 
is unrealistic. 

As measured in the excito-repellency test (Char- 
eon viriyaphap et al. 1997), noncontact repellency is 
not as quick or pronounced a behavioral response 
as contact irritancy. In the field, most mosquitoes 
stimulated to prematurely exit houses will probably 
do so only after physical contact with insecticide 
residues. However, the mosquito must first enter the 
house before it can make contact with insecticide. 
Except for a specimen that enters and then exits the 
house in pursuit of a host, the stimuli for the 3 
behavioral acts of entering, resting indoors, and 
eventually exiting the house are different. 

Noncontact repellency probably exerts its most 
powerful influence by preventing mosquitoes from 
entering houses. This repellency action has been 
documented in several field studies against several 
vector species (Roberts and Andre 1994). As ex- 
amples, Roberts and Alecrim (1991) showed with 
experimental houses that Anopheles darlingi Root 
females practically stopped entering a house after 
it was sprayed with DDT, approaching a 100% re- 
duction in indoor biting. Smith and Webley (1968) 
showed a 60-70% reduction in house entering by 
Anopheles gambiae Giles females after an experi- 
mental house was sprayed with DDT. Shalaby 
(1966) showed that, in comparison with a control 



5 STATA* statistical software was provided by Stata 
Corporation, 702 University Drive East, College Station, 
TX 77840. 



house, 75% fewer Anopheles culicifacies Giles 
specimens were collected inside a DDT-sprayed 
house, even with all surfaces screened to preclude 
physical contact with DDT. With this background, 
it is clear that the excito-repellency test may pro- 
vide a direct measure of vector responsiveness to 
the irritant affects of insecticides, and the results 
might be predictive of actions inside of houses un- 
der field conditions. However, the excito-repellency 
test is perhaps less informative of insecticidal im- 
pact on the act of entering a sprayed house. 

Behavioral responses of vectors to insecticides 
are important, but generally neglected areas of 
study. Progress in understanding the importance of 
insecticide avoidance behaviors has been impeded 
by the lack of acceptable test systems. To date, no 
WHO-recommended test methods discriminate be- 
tween contact versus noncontact insecticide-in- 
duced behaviors. No tests are easily conducted or, 
excluding the excito-repellency test box, provide a 
powerful and reproducible result. Additionally, no 
test data are amenable to sophisticated statistical 
analyses. The excito-repellency test box, the test, 
and data analysis methods (test system) described 
in this report were designed to resolve some of the 
problems identified by Roberts et al. (1984). The 
test system has already been used in an extensive 
study of behavioral responses of different Anoph- 
eles albimanus Wiedemann populations to DDT, 
permethrin, and deltamethrin (Chareonviriyaphap 
et al. 1997). Using this test system in combination 
with susceptibility, isozyme, and esterase tests, 
Chareonviriyaphap et al. (1997) obtained no evi- 
dence of relationships between physiological resis- 
tance and behavioral responses of An. albimanus 
females to insecticides. 

The excito-repellency test system described in 
this report offers the following desirable attributes: 

1 . Exposure chambers are constructed of metal and 
can be chemically cleaned for use with different 
doses and types of insecticides. 

2. Screened inserts provide a capability to test be- 
havioral responses without physical contact with 
insecticide. 

3. Mosquitoes are easily transferred to the expo- 
sure chambers and are easily removed from the 
chambers after the test is complete. 

4. Counts by 1-min intervals are sensitive to rapid 
behavioral responses to insecticides. 

5. Highly reproducible test results are obtained 
(Chareonviriyaphap et al. 1997). 

6. The survival analysis method is a robust treat- 
ment of data that minimizes the loss of infor- 
mation. 

7. Comparative summary statistics in the form of 
escape times for 50 and 90% (ET 50 and EX*,) of 
test specimens to escape exposure chambers can 
be estimated. 

8. Specimens that escape at different time intervals 
and specimens that remain in the exposure 



March 1997 



Excito-Repellency Testing 



17 



chamber throughout the test can be held and 
scored for 24-h mortalities. 

In our studies (Chareonviriyaphap et al. 1997), 
we employed test papers that were prepared ac- 
cording to WHO specifications by the United States 
Army Center for Health Promotion and Preventive 
Medicine, Aberdeen Proving Ground, MD. Test pa- 
pers are also available from the WHO. 

ACKNOWLEDGMENT 

This research was supported by an intramural 
grant (R087EK) from the Uniformed Services Uni- 
versity of the Health Sciences, Befhesda, MD. 

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